Economical approaches to genetically modify or disrupt the expression of genes in rats would be widely applicable to accelerating scientific discovery. In view of this potential, we previously established protocols for isolating, propagating, genetically modifying and determining the germline transmission rates of rat spermatogonial cultures as a newly applied type of germline stem cell. We initially chose the rat as a species for these studies due to its popularity as a laboratory scale mammalian model for human disease. This is based on the need for new protocols to mutate genes in rats to study biological processes that are not as robustly modeled using mice. In pilot studies, Sleeping Beauty gene-trap transposons were successfully used to disrupt transcribed sequences in spermatogonial lines. Accordingly, Specific Aims of the current project will exploit a new and robust "sterile-testis-complementation model" in which donor spermatogonia and sterile recipients serve as vectors to most effectively transmit donor transgenes through the rat germline. This system maximizes germline transmission of mutatant genes directly from rat spermatogonia, and therefore, by-passes the intermediate need to produce and breed mosaic/chlmairic progeny. In Specific Aim 1, germline transmission rates will be defined for different commonly applied selectable markers after their integration into rat spermatogonial genomes using transposons (i.e. b-Geo, Venus, dtTomato). In Specific Aim 2, mutational spectra of transcribed genes will be determined in both, cultures of rat spermatogonia, plus live rats produced with the spermatogonial cultures, using two different transposon systems (i.e. Sleeping Beauty &PiggyBac) designed to generate conditional and reversible knockout rats. In specific Aim 3, large numbers of gene knockouts within rat spermatogonial libraries will be fully annotated using whole genome sequencing approaches. The library will be screened for the chosen targeted germlines, which will then be used to generate the desired mutant rats. Thus, pre-defining conditional gene-trap mutations within complex genomic libraries of mutant spermatogonia will enable the most user friendly and cost-effective strategy available to target the disruption genes in the rat. PUBLIC HEALTH RELEVANCE (provided by applicant): This project will use contemporary technologies to build an unprecedented resource of rat genetic models for the scientific community. The resource of will dramatically expand the biomedical potential and discovery in essentially every field of the life sciences by allowing researchers to study relationships between gene function and human health using the rat as a genetic model.